Novel human phosphatase inhibitor protein

ABSTRACT

The present invention provides a novel human phosphatase inhibitor protein (HPIP) and polynucleotides which identify and encode HPIP. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding HPIP and a method for producing HPIP. The invention also provides for agonists, antibodies, or antagonists specifically binding HPIP, and their use, in the prevention and treatment of diseases associated with expression of HPIP. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding HPIP for the treatment of diseases associated with the expression of HPIP. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding HPIP.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/262,610, filed Mar. 4, 1999, which is a divisionalapplication of U.S. application Ser. No. 08/766,738, filed Dec. 13,1996, now U.S. Pat. No. 5,916,749, the contents all of which are herebyincorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a novel human phosphatase inhibitor protein and to the use of thesesequences in the diagnosis, prevention, and treatment of cancer, AIDS,immunodeficiencies, autoimmune diseases, inflammatory diseases, andproliferative diseases.

BACKGROUND OF THE INVENTION

[0003] Major histocompatibility complex (MHC) class II molecule is anessential component of the immune response to foreign antigens. The MHCclass II molecule is expressed on the surface of B lymphocytes,macrophages, dendritic cells, and activated T cells (Kaufman, J. F. etal. (1984) Cell 36:1-13). The MHC class II molecule presents a processedforeign antigen at the cell surface to receptors located on competenthelper T cells (Lanzavecchia, A. (1985) Nature 314:537-539). Antigensare processed (proteolytically degraded into small peptides) and loadedonto the MHC class II molecules in an acidic, hydrolase-richlysosome-like cellular compartment prior to transport to the cellsurface (Peters, P. J. et al. (1991) Nature 349:669-676). Binding of theMHC class II/antigen complex to the receptor leads to activation of thehelper T cells.

[0004] MHC class II molecules are heterodimeric glycoproteins composedof an α- and β-chain (Kaufman, J. F. et al., supra). Binding of anextracellular region of the MHC class II dimer by T cell receptors inthe presence of processed antigen, or by anti-MHC antibodies, is thoughtto transduce a signal into the cell across the plasma membrane.Cross-linking of MHC class II molecules leads to increased intracellularcAMP levels and translocation of protein kinase C (PKC) to the nucleusin mouse cells (Cambier, J. C. et al. (1987) Nature 327:629-632), and totyrosine phosphorylation, phosphatidylinositol turnover, increased freeintracellular Ca²⁺, and PKC activation in human cells (Mooney, N. A. etal. (1990) J. Immunol. 145:2070-2076; Brick-Ghannam, C. et al. (1991) J.Biol. Chem. 266:24169-24175).

[0005] Signal transduction by cross-linked MHC class II moleculesimplies that an intracellular portion of the dimer plays an essentialrole and thereby contributes to activation of B cells and helper T cellsduring the T cell-dependent immune response. Experiments with cell linesexpressing truncated forms of the mouse MHC class II molecule supportthis model and suggest that soluble cytoplasmic proteins interactingwith the intracellular domain of the dimer participate in signaltransduction (Ostrand-Rosenberg, S. et al. (1991) J. Immunol.147:2419-2422).

[0006] A search for cytoplasmic proteins that interact with theintracellular domain of MHC class II molecules detects two proteins fromseparate genes that are designated PHAPI and PHAPII. These proteinsinteract specifically with the intracellular domain of the MHC class IIα-chain, but not with the β-chain or other unrelated proteins (Vaesen,M. et al. (1994) Biol. Chem. Hoppe-Seyler 375:113-126). Sequenceanalyses indicate that carboxy ends of PHAPI and PHAPII are highlyacidic and are similar to the acidic activating domains of transcriptionfactors (Vaesen, M. et al., supra; Ptashne, M. (1988) Nature335:683-689).

[0007] PHAPI has two regularly spaced leucine/isoleucine motifs in theamino terminal region of the protein. These motifs are similar to thosefound in yeast adenylyl cyclase, human platelet receptor protein, andyeast mitosis dephosphorylation regulator (Vaesen, M. et al., supra; Li,M. et al. (1996) Biochemistry 35:6998-7002). PHAPI also has a nuclearlocalization signal in its carboxy terminus and significant amounts aredetected both in the nucleus and diffusely distributed throughout thecytoplasm (Vaesen, M. et al., supra).

[0008] Additional analysis shows that PHAPI is identical to I₁ ^(PP2)A,a specific and potent inhibitor of the protein phosphatase 2A (PP2A; Li,M. et al., supra). PP2A is an important protein serine/threoninephosphatase that is involved in the regulation of diverse mammalian cellprocesses including reentry of quiescent cells into the cell cycle(Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508; Shenolikar, S. and A.C. Nairn (1991) Adv. Second Messenger Phosphoprot. Res. 23:1-121; Mumby,M. C. and G. Walter (1993) Physiol. Rev. 73:673-699).

[0009] The discovery of polynucleotides encoding the human phosphataseinhibitor protein, and the molecules themselves, provides a means toinvestigate signal transduction, mitogenesis and cellular proliferation,and the immune response. Discovery of molecules related to phosphataseinhibitor protein satisfies a need in the art by providing newdiagnostic or therapeutic compositions useful in the diagnosis,prevention, and treatment of cancer, AIDS, immunodeficiencies,autoimmune diseases, inflammatory diseases, and proliferative diseases.

SUMMARY OF THE INVENTION

[0010] The present invention features a novel human phosphataseinhibitor protein hereinafter designated HPIP and characterized ashaving similarity to PHAPI (I₁ ^(PP2)A).

[0011] Accordingly, the invention features a substantially purified HPIPhaving the amino acid sequence shown in SEQ ID NO:1.

[0012] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode HPIP. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

[0013] The invention also relates to a polynucleotide sequencecomprising the complement of SEQ ID NO:2 or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO:2.

[0014] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode HPIP. Thepresent invention also features antibodies which bind specifically toHPIP, and pharmaceutical compositions comprising substantially purifiedHPIP. The invention also features the use of agonists and antagonists ofHPIP.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIGS. 1A, 1B, and 1C shows the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of HPIP. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.,Ltd., San Bruno, Calif.).

[0016]FIGS. 2A, 2B, and 2C shows the amino acid sequence alignmentsamong HPIP (SEQ ID NO:1), HSPHAPI2A (G1498225; SEQ ID NO:3), and PHAPI(G403007; SEQ ID NO:4). The alignment was produced using themultisequence alignment program of DNASTAR software (DNASTAR Inc,Madison Wis.).

[0017]FIG. 3 shows the hydrophobicity plot (MACDNASIS PRO software) forHPIP, SEQ ID NO:1; the positive X axis reflects amino acid position, andthe negative Y axis, hydrophobicity.

[0018]FIG. 4 shows the hydrophobicity plot for PHAPI, SEQ ID NO:4.

[0019]FIGS. 5A and 5B shows the northern analysis for SEQ ID NO:2. Thenorthern analysis was produced electronically using the LIFESEQ™database (Incyte Pharmaceuticals, Inc., Palo Alto, Calif.).

DESCRIPTION OF THE INVENTION

[0020] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0021] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0022] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0023] Definitions

[0024] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide or protein sequence, and fragments orportions thereof, and to naturally occurring or synthetic molecules.

[0025] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0026] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0027] HPIP, as used herein, refers to the amino acid sequences ofsubstantially purified HPIP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic or recombinant.

[0028] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using XL-PCR kit (Perkin Elmer, Norwalk, Conn.) in the 5′and/or the 3′ direction and resequenced, or which has been assembledfrom the overlapping sequences of more than one Incyte clone using theGELVIEW fragment assembly system (GCG, Madison, Wis,), or which has beenboth extended and assembled.

[0029] A “variant” of HPIP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0030] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0031] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0032] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0033] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic HPIP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0034] The term “agonist”, as used herein, refers to a molecule which,when bound to HPIP, causes a change in HPIP which modulates the activityof HPIP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to HPIP.

[0035] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to HPIP, blocks or modulates the biologicalor immunological activity of HPIP. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to HPIP.

[0036] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of HPIP. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of HPIP.

[0037] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of HPIPor portions thereof and, as such, is able to effect some or all of theactions of PHAPI-like molecules.

[0038] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding HPIP or the encoded HPIP.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0039] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0040] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0041] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0042] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen binds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., Cot or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0043] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, for thesequence “A-G-T” binds to the complementary sequence “T-C-A”.Complementarity between two single-stranded molecules may be “partial”,in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands.

[0044] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0045] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0046] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm−5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0047] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0048] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length human HPIP and fragments thereof.

[0049] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0050] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0051] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0052] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding HPIPor fragments thereof may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0053] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding HPIP in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0054] “Alterations” in the polynucleotide of SEQ ID NO:2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding HPIP including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes HPIP (e.g., by alterations in the pattern of restrictionfragment length polymorphisms capable of hybridizing to SEQ ID NO:2),the inability of a selected fragment of SEQ ID NO:2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding HPIP (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

[0055] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind HPIPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0056] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0057] The Invention

[0058] The invention is based on the discovery of a novel humanphosphatase inhibitor protein (HPIP), the polynucleotides encoding HPIP,and the use of these compositions for the diagnosis, prevention ortreatment of cancer, AIDS, immunodeficiencies, autoimmune diseases,inflammatory diseases, and proliferative diseases.

[0059] Nucleic acids encoding the HPIP of the present invention werefirst identified in Incyte Clone 1813361 from the prostate tumor cDNAlibrary (PROSTUT12) through a computer-generated search for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 1813361 (PROSTUT12), 857548 (NGANNOT01), 1603390 (LUNGNOT15),686145 (UTRSNOT02), 790139 (PROSNOT03), and 184875 (CARDNOT01).

[0060] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, and 1C. HPIP is 251 amino acids in length and has two potentialN-linked glycosylation sites at residues 89 and 94. HPIP has twopotential tyrosine phosphorylation sites at residues 131 and 148. It hasa potential nuclear localization signal at residues 236-241, and twoleucine/isoleucine motifs at residues 54-71 and 100-120. HPIP haschemical and structural homology with HSPHAPI2A (G1498225; SEQ ID NO:3),and PHAPI (G403007; SEQ ID NO:4). In particular, HPIP shares 98% and 67%identity with HSPHAPI2A and PHAPI, respectively (FIGS. 2A and 2B). Asillustrated by FIGS. 3 and 4, HPIP and PHAPI have similar hydrophobicityplots. HPIP, HSPHAPI2A, and PHAPI all have very acidic carboxy domainsand isoelectric points of 3.76, 3.77, and 3.83, respectively. Northernanalysis (FIGS. 5A and 5B) shows the expression of HPIP in various cDNAlibraries. Approximately 37% of these libraries are from tumors andimmortalized cell lines; 14% of the libraries are from cells of theimmune system and diseased tissue likely to be associated with increasednumbers of lymphocytes.

[0061] The invention also encompasses HPIP variants. A preferred HPIPvariant is one having at least 80%, and more preferably 90%, amino acidsequence similarity to the HPIP amino acid sequence (SEQ ID NO:1). Amost preferred HPIP variant is one having at least 95% amino acidsequence similarity to SEQ ID NO:1.

[0062] The invention also encompasses polynucleotides which encode HPIP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of HPIP can be used to generate recombinant molecules whichexpress HPIP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B, and 1C.

[0063] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding HPIP, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring HPIP, and all such variations are to beconsidered as being specifically disclosed.

[0064] Although nucleotide sequences which encode HPIP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HPIP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HPIP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HPIP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0065] The invention also encompasses production of DNA sequences, orportions thereof, which encode HPIP and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding HPIP or any portionthereof.

[0066] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0067] Altered nucleic acid sequences encoding HPIP which areencompassed by the invention include deletions, insertions orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent HPIP. The encodedprotein may also contain deletions, insertions or substitutions of aminoacid residues which produce a silent change and result in a functionallyequivalent HPIP. Deliberate amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of HPIP is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

[0068] Also included within the scope of the present invention arealleles of the genes encoding HPIP. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0069] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE DNA polymerase (US Biochemical Corp,Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7polymerase (Amersham, Chicago, Ill.), or combinations of recombinantpolymerases and proofreading exonucleases such as the ELONGASEamplification system (GIBCOIBRL, Gaithersburg, Md.). Preferably, theprocess is automated with machines such as the Hamilton MICROLAB 2200(Hamilton, Reno, Nev.), Peltier thermal cycler (PTC200; M.J. Research,Watertown, Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

[0070] The nucleic acid sequences encoding HPIP may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0071] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nuc. Acids Res. 16:8186). The primers may be designed using OLIGO 4.06after “4.06” (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0072] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0073] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nuc. Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. When screening for full-length cDNAs, it ispreferable to use libraries that have been size-selected to includelarger cDNAs. Also, random-primed libraries are preferable, in that theywill contain more sequences which contain the 5′ regions of genes. Useof a randomly primed library may be especially preferable for situationsin which an oligo d(T) library does not yield a full-length cDNA.Genomic libraries may be useful for extension of sequence into the 5′and 3′ non-transcribed regulatory regions.

[0074] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0075] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode HPIP, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of HPIP in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressHPIP.

[0076] As will be understood by those of skill in the art, it may beadvantageous to produce HPIP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0077] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterHPIP encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0078] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HPIP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HPIP activity, it may be useful toencode a chimeric HPIP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HPIP encoding sequence and theheterologous protein sequence, so that HPIP may be cleaved and purifiedaway from the heterologous moiety.

[0079] In another embodiment, sequences encoding HPIP may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nuc. Acids Res. Symp. Ser.7:215-223; Horn, T. et al. (1980) Nuc. Acids Res. Symp. Ser. 7:225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of HPIP, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431Apeptide synthesizer (Perkin Elmer).

[0080] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, T., supra). Additionally, the aminoacid sequence of HPIP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0081] In order to express a biologically active HPIP, the nucleotidesequences encoding HPIP or functional equivalents, may be inserted intoan appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0082] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding HPIPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0083] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding HPIP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0084] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding HPIP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0085] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for HPIP. For example, whenlarge quantities of HPIP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asthe BLUESCRIPT phagemid (Stratagene), in which the sequence encodingHPIP may be ligated into the vector in frame with sequences for theamino-terminal Met and the subsequent 7 residues of β-galactosidase sothat a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. PGEXvectors (Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0086] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0087] In cases where plant expression vectors are used, the expressionof sequences encoding HPIP may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0088] An insect system may also be used to express HPIP. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding HPIPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of HPIP will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which HPIP may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0089] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding HPIP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing HPIP in infected host cells (Logan, J.and T. Shenk. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0090] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding HPIP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding HPIP, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0091] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0092] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress HPIP may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0093] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0094] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding HPIP isinserted within a marker gene sequence, recombinant cells containingsequences encoding HPIP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding HPIP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0095] Alternatively, host cells which contain the nucleic acid sequenceencoding HPIP and express HPIP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0096] The presence of polynucleotide sequences encoding HPIP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding HPIP.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding HPIP todetect transformants containing DNA or RNA encoding HPIP. As used herein“oligonucleotides” or “oligomers” refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20-25nucleotides, which can be used as a probe or amplimer.

[0097] A variety of protocols for detecting and measuring the expressionof HPIP, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson HPIP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0098] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding HPIPinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding HPIP, or any portions thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio). Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0099] Host cells transformed with nucleotide sequences encoding HPIPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode HPIP may be designed to contain signal sequences which directsecretion of HPIP through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encodingHPIP to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and HPIP may be used to facilitate purification. Onesuch expression vector provides for expression of a fusion proteincontaining HPIP and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography as described in Porath, J. et al. (1992, Prot.Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides ameans for purifying HPIP from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

[0100] In addition to recombinant production, fragments of HPIP may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431Apeptide synthesizer (Perkin Elmer). Various fragments of HPIP may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0101] Therapeutics

[0102] Based on the chemical and structural homology among HPIP (SEQ IDNO:1), HSPHAPI2A (SEQ ID NO:3), and PHAPI (I₁ ^(PP2A)) (SEQ ID NO:4),HPIP appears to play a role in the development of diseases resultingfrom excessive cellular growth and division which include, but notlimited to, cancer, autoimmune diseases, inflammatory diseases,proliferative diseases, and diseases resulting from insufficientcellular growth and division which include, but not limited to, AIDS andother infectious or inherited immunodeficiencies.

[0103] Therefore, in one embodiment, HPIP, or a fragment or derivativethereof, may be administered to a subject to prevent or treat cancer.Such conditions and diseases include, but are not limited to, leukemia,and cancer of the stomach, kidney, ovary, prostate, bladder, colon,lung, brain, breast, and thyroid.

[0104] In another embodiment, HPIP, or a fragment or derivative thereof,may be administered to a subject to prevent or treat autoimmunediseases. Such autoimmune diseases include, but are not limited to,systemic lupus, myasthenia gravis, multiple sclerosis, rheumatoidarthritis, Sjögren's syndrome, Grave's disease, autoimmune thyroiditis,diabetes, pancreatitis; ulcerative colitis, Crohn's disease, andatrophic gastritis.

[0105] In another embodiment, HPIP, or a fragment or derivative thereof,may be administered to a subject to prevent or treat inflammatorydiseases which include, but are not limited to, osteoarthritis, asthma,and inflammatory bowel disease.

[0106] In another embodiment, HPIP, or a fragment or derivative thereof,may be administered to a subject to prevent or treat proliferativediseases which include, but are not limited to prostate hypertrophy,atherosclerosis, restenosis, psoriasis, and lymphadenopathy.

[0107] In another embodiment, a vector capable of expressing HPIP, or afragment or a derivative thereof, may be administered to a subject toprevent or treat cancer, which include, but are not limited to, thecancers listed above.

[0108] In another embodiment, a vector capable of expressing HPIP, or afragment or a derivative thereof, may be administered to a subject toprevent or treat autoimmune diseases, which include, but not limited to,those listed above.

[0109] In another embodiment, a vector capable of expressing HPIP, or afragment or a derivative thereof, may be administered to a subject toprevent or treat inflammatory diseases which include, but not limitedto, those listed above.

[0110] In another embodiment, a vector capable of expressing HPIP, or afragment or a derivative thereof, may be administered to a subject toprevent or treat proliferative diseases which include, but not limitedto, those listed above.

[0111] In another aspect, agonists which are specific for HPIP may beadministered to a subject to prevent or treat diseases resulting fromexcessive cell growth and division, which include, but not limited to,those listed above.

[0112] In one embodiment, antagonists or inhibitors of HPIP may beadministered to a subject to treat or prevent immunodeficiencies whichinclude, but are not limited to, AIDS and other infectious or inheritedimmunodeficiencies.

[0113] In one aspect, antibodies which are specific for HPIP may be useddirectly as an antagonist, or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress HPIP.

[0114] In another embodiment, a vector expressing antisense of thepolynucleotide encoding HPIP may be administered to a subject to preventor treat immunodeficiencies which include, but are not limited to, AIDSand other infectious or inherited immunodeficiencies.

[0115] In other embodiments, any of the therapeutic proteins,antibodies, agonists, antagonists, antisense sequences or vectorsdescribed above may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent.

[0116] Antagonists or inhibitors of HPIP may be produced using methodswhich are generally known in the art. In particular, purified HPIP maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind HPIP.

[0117] Antibodies which are specific for HPIP may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express HPIP.The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0118] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith HPIP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0119] It is preferred that the peptides, fragments or oligopeptidesused to induce antibodies to HPIP have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of HPIP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0120] Monoclonal antibodies to HPIP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0121] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceHPIP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0122] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0123] Antibody fragments which contain specific binding sites for HIPmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0124] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between HPIP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HPIP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0125] In another embodiment of the invention, the polynucleotidesencoding HPIP, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding HPIP may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding HPIP. Thus, antisense molecules may be used to modulate HPIPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding HPIP.

[0126] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingHPIP. These techniques are described both in Sambrook et al. (supra) andin Ausubel et al. (supra).

[0127] Genes encoding HPIP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes HPIP. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0128] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding HPIP, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0129] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding HPIP.

[0130] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0131] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding HPIP. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0132] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′-O-methyl phosphodiesterlinkages within the backbone of the molecule. This concept is inherentin the production of PNAs and can be extended in all of these moleculesby the inclusion of nontraditional bases such as inosine, queosine, andwybutosine, as well as acetyl-, methyl-, thio-, and similarly modifiedforms of adenine, cytidine, guanine, thymine, and uridine which are notas easily recognized by endogenous endonucleases.

[0133] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0134] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0135] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HPIP,antibodies to HPIP, mimetics, agonists, antagonists, or inhibitors ofHPIP. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0136] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0137] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0138] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0139] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0140] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0141] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0142] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0143] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0144] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0145] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0146] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of HPIP, such labeling wouldinclude amount, frequency, and method of administration.

[0147] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0148] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0149] A therapeutically effective dose refers to that amount of activeingredient, for example HPIP or fragments thereof, antibodies of HPIP,agonists, antagonists or inhibitors of HPIP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0150] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0151] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0152] Diagnostics

[0153] In another embodiment, antibodies which specifically bind HPIPmay be used for the diagnosis of conditions or diseases characterized byexpression of HPIP, or in assays to monitor patients being treated withHPIP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for HPIP includemethods which utilize the antibody and a label to detect HPIP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0154] A variety of protocols including ELISA, RIA, and FACS formeasuring HPIP are known in the art and provide a basis for diagnosingaltered or abnormal levels of HPIP expression. Normal or standard valuesfor HPIP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to HPIP under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of HPIPexpressed in subject, control and disease, samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0155] In another embodiment of the invention, the polynucleotidesencoding HPIP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHPIP may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of HPIP,and to monitor regulation of HPIP levels during therapeuticintervention.

[0156] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HPIP or closely related molecules, may be used to identifynucleic acid sequences which encode HPIP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding HPIP, alleles, or related sequences.

[0157] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the HPIP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring HPIP.

[0158] Means for producing specific hybridization probes for DNAsencoding HPIP include the cloning of nucleic acid sequences encodingHPIP or HPIP derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0159] Polynucleotide sequences encoding HPIP may be used for thediagnosis of conditions or diseases which are associated with expressionof HPIP. Examples of such conditions or diseases include diseasesresulting from excessive cellular growth and division including, but notlimited to, cancer, autoimmune diseases, inflammatory diseases,proliferative diseases, and diseases resulting from insufficientcellular growth and division including, but not limited to, AIDS andother infectious or inherited immunodeficiencies. The polynucleotidesequences encoding HPIP may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; orin dip stick, pin, ELISA or chip assays utilizing fluids or tissues frompatient biopsies to detect altered HPIP expression. Such qualitative orquantitative methods are well known in the art.

[0160] In a particular aspect, the nucleotide sequences encoding HPIPmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding HPIP may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding HPIP in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0161] In order to provide a basis for the diagnosis of diseaseassociated with expression of HPIP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes HPIP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0162] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0163] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0164] Additional diagnostic uses for oligonucleotides designed from thesequences encoding HPIP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′→3′) and another with antisense(3′←5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

[0165] Methods which may also be used to quantitate the expression ofHPIP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

[0166] In another embodiment of the invention, the nucleic acidsequences which encode HPIP may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0167] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding HPIP on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0168] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0169] In another embodiment of the invention, HPIP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenHPIP and the agent being tested, may be measured.

[0170] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to HPIP large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with HPIP, or fragments thereof, and washed. Bound HPIP is thendetected by methods well known in the art. Purified HPIP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0171] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding HPIPspecifically compete with a test compound for binding HPIP. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with HPIP.

[0172] In additional embodiments, the nucleotide sequences which encodeHPIP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0173] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0174] I Prostut12 cDNA Library Construction

[0175] The PROSTUT12 cDNA library was constructed from prostate tumortissue obtained from a 65-year-old Caucasian male during a radicalprostatectomy. The patient presented with elevated prostate-specificantigen and was diagnosed as having a malignant neoplasm of theprostate. The pathology of the tumor indicated that the predominant massinvolving the right anterior prostate peripherally was an adenocarcinoma(Gleason grade 2+2). Multiple microscopic foci were identified in theleft and right sides of the prostate but did not involve the capsule.Perineural invasion was present. Multiple pelvic lymph nodes werenegative for tumor.

[0176] The frozen tissue was homogenized and lysed using a BrinkmannPOLYTRON homogenizer PT-3000 (Brinkmann Instruments, Westbury, N.Y.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using a Beckman SW28 rotor in a Beckman L8-70Multracentrifuge (Beckman Instruments, Fullerton, Calif.) for 18 hours at25,000 rpm at ambient temperature. The RNA was extracted with acidphenol, pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumesof ethanol, resuspended in RNase-free water, and DNase-treated at 37° C.The RNA extraction was repeated with acid phenol, pH 4.7, and the RNAprecipitated with sodium acetate and ethanol, as above. The mRNA wasisolated using the OLIGOTEX kit (QIAGEN, Inc., Chatsworth, Calif.) andused to construct the cDNA library.

[0177] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT plasmid system (Cat. #18248-013, Gibco BRL). A newplasmid was constructed using the following procedures: The commercialplasmid PSPORT 1 (Gibco BRL) was digested with Eco RI restriction enzyme(New England Biolabs, Beverley, Mass.), the overhanging ends of theplasmid were filled with Klenow enzyme (New England Biolabs) and2′-deoxynucleotide-5′-triphosphates (dNTPs); the intermediate plasmidwas self-ligated and transformed into the bacterial host, E. coli strainJM109.

[0178] Quantities of this intermediate plasmid were digested with HindIII restriction enzyme (New England Biolabs), the overhanging ends werefilled with Klenow and dNTPs, and a 10-mer linker of sequence 5′ . . .CGGAATTCCG . . . 3′ was phosphorylated and ligated onto the blunt ends.The product of the ligation reaction was digested with EcoRI andself-ligated. Following transformation into JM109 host cells, plasmidsdesignated pINCY were isolated and tested for the ability to incorporatecDNAs using Not I and Eco RI restriction enzymes.

[0179] PROSTUT12 cDNAs were fractionated on a SEPHAROSE CL4B column(Cat. #275105-01, Pharmacia), and those cDNAs exceeding 400 bp wereligated into pINCY I. The plasmid pINCY I was subsequently transformedinto DH5α™ competent cells (Cat. #18258-012, Gibco BRL).

[0180] II. Isolation and Sequencing of cDNA Clones

[0181] Plasmid DNA was released from the cells and purified using theR.E.A.L. PREP-96 plasmid kit (Cat. #26173, QIAGEN, Inc.). This kitenabled the simultaneous purification of 96 samples in a 96-well blockusing multi-channel reagent dispensers. The recommended protocol wasemployed except for the following changes: 1) the bacteria were culturedin 1 ml of sterile Terrific Broth (Cat. #22711, LIFE TECHNOLOGIES™) withcarbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, thecultures were incubated for 19 hours and at the end of incubation, thecells were lysed with 0.3 ml of lysis buffer; and 3) followingisopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1ml of distilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

[0182] The cDNAs were sequenced by the method of Sanger et al. (1975, J.Mol. Biol. 94:441f), using a Hamilton MICROLAB 2200 (Hamilton, Reno,Nev.) in combination with Peltier thermal cyclers (PTC200 from M. J.Research, Watertown, Mass.) and Applied Biosystems 377 DNA sequencingsystems; and the reading frame was determined.

[0183] III Homology Searching of cDNA Clones and their Deduced Proteins

[0184] Each cDNA was compared to sequences in GenBank using a searchalgorithm developed by Applied Biosystems and incorporated into theINHERIT-670 sequence analysis system. In this algorithm, PatternSpecification Language (TRW Inc, Los Angeles, Calif.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

[0185] Peptide and protein sequence homologies were ascertained usingthe INHERIT-670 sequence analysis system using the methods similar tothose used in DNA sequence homologies. Pattern Specification Languageand parameter windows were used to search protein databases forsequences containing regions of homology which were scored with aninitial value. Dot-matrix homology plots were examined to distinguishregions of significant homology from chance matches.

[0186] BLAST, which stands for Basic Local Alignment Search Tool(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990)J. Mol. Biol. 215:403-410), was used to search for local sequencealignments. BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. BLAST is useful for matches which donot contain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

[0187] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0188] IV Northern Analysis

[0189] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0190] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0191] The basis of the search is the product score which is defined as:

% sequence identity×% maximum BLAST score/100

[0192] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0193] The results of northern analysis are reported as a list oflibraries in which the transcript encoding HPIP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0194] V Extension of HPIP-Encoding Polynucleotides to Full Length or toRecover Regulatory Sequences

[0195] Full length HPIP-encoding nucleic acid sequence (SEQ ID NO:2) isused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3+, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

[0196] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0197] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier thermal cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0198] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK DNA purification kit (QIAGEN Inc.,Chatsworth, Calif.). After recovery of the DNA, Klenow enzyme is used totrim single-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0199] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 ul of each sample istransferred into a PCR array.

[0200] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0201] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0202] VI Labeling and Use of Hybridization Probes

[0203] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 primer analysis software (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 μCi of [γ-³²P]adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN®, Boston, Mass.). The labeled oligonucleotides are substantiallypurified with SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn).A portion containing 10⁷ counts per minute of each of the sense andantisense oligonucleotides is used in a typical membrane basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II;DuPont NEN®).

[0204] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (NYTRAN PLUS membrane, Schleicher& Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at40° C. To remove nonspecific signals, blots are sequentially washed atroom temperature under increasingly stringent conditions up to0.1×saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMATAR autoradiography film (Kodak, Rochester, N.Y.) is exposed to theblots, or the blots are placed in a PHOSPHOIMAGER cassette (MolecularDynamics, Sunnyvale, Calif.) for several hours, hybridization patternsare compared visually.

[0205] VII Antisense Molecules

[0206] Antisense molecules to the HPIP-encoding sequence, or any partthereof, is used to inhibit in vivo or in vitro expression of naturallyoccurring HPIP. Although use of antisense oligonucleotides, comprisingabout 20 base-pairs, is specifically described, essentially the sameprocedure is used with larger cDNA fragments. An oligonucleotide basedon the coding sequences of HPIP, as shown in FIGS. 1A, 1B, and 1C isused to inhibit expression of naturally occurring HPIP. Thecomplementary oligonucleotide is designed from the most unique 5′sequence as shown in FIGS. 1A, 1B, and 1C used either to inhibittranscription by preventing promoter binding to the upstreamnontranslated sequence or translation of an HPIP-encoding transcript bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 53 sequence of SEQ ID NO:2, an effective antisenseoligonucleotide includes any 15-20 nucleotides spanning the region whichtranslates into the signal or 5′ coding sequence of the polypeptide asshown in FIGS. 1A, 1B, and 1C.

[0207] VIII Expression of HPIP

[0208] Expression of HPIP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express HPIP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0209] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of 13-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of HPIP into the bacterial growth media which can be useddirectly in the following assay for activity.

[0210] IX Demonstration of HPIP Activity

[0211] HPIP cDNA is ligated into the pET-3d plasmid (Novagen, Madison,Wis.), linearized with the appropriate restriction enzyme(s), in orderto place HPIP expression under the control of the bacterial lacZpromoter contained in the plasmid. E. coli BL21(DE3)pLysS (Novagen) istransformed with a portion of the ligation mixture under the conditionsspecified by the manufacturer. A colony containing the desired plasmidis isolated and grown up to log phase at 37° C. Synthesis of HPIP isinduced by the addition of IPTG (GIBCO BRL) to the medium (0.5 mM, finalconcentration) and incubation for two additional hours.

[0212] HPIP is isolated from the IPTG-induced cells and purifiedessentially as described by Li et al. (1996; supra) for I₁ ^(PP2A)(PHAPI). In brief, the cells are lysed in a French press at 1200 psiunder suitable conditions of pH and ionic strength in the presence ofprotease inhibitors such as 1 mM benzamidine and 0.1 mMphenylmethylsulfonyl fluoride (Sigma, St. Louis, Mo.). The lysate isclarified by centrifugation. HPIP is purified by a series of steps whichmay include chromatography on poly(L-lysine)-agarose, Sephacryl-200(Pharmacia Biotech), affinity chromatography on agarose attached to asynthetic peptide equivalent to the carboxy terminus of MHC class IIα-chain, and acid precipitations (Li, M. et al., supra; Vaesen, M. etal., supra). Purification of the HPIP is monitored at each step bySDS-polyacrylamide gel electrophoresis using techniques well known inthe art.

[0213] Bovine kidney PP2A is purified to homogeneity using thetechniques described by Amick et al. (1992; Biochem. J. 287:1019-1022).Bovine brain myelin basic protein (MBP) is purified as described byDeibler et al. (1984; Prog. Clin. Biol. Res. 146:249-256) andphosphorylated with [γ-³²P]ATP as previously described (Li, M. et al.(1995) Biochemistry 34:1988-1996).

[0214] Isolated HPIP is assayed for inhibition of protein phosphatasePP2A activity. A constant amount of PP2A (ca. 0.005 unit; 0.1 ng) isincubated with a fixed amount of [³²P]-labeled MBP in the presence ofincreasing amounts of HPIP. After incubation under appropriateconditions of time, temperature, pH, and ionic strength (Li, .M. et al.(1995), supra), the proteins are precipitated with cold trichloroaceticacid and collected on nitrocellulose filters with a 0.45 mμ pore size(Millipore, Bedford, Mass.). The filters are dried and immersed in acommercially available scintillation fluid prior to counting in ascintillation counter (Beckman). The amount of [³²P]phosphate releasedfrom MBP by PP2A in the absence of HPIP is set as 100% phosphataseactivity, and the percent inhibition of PP2A activity versusconcentration of HPIP is calculated. Increasing amounts of HPIP willprevent the release of [³²P]phosphate from MBP by inhibiting PP2Aactivity.

[0215] X Production of HPIP Specific Antibodies

[0216] HPIP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0217] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) byreaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS;Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLHcomplex in complete Freund's adjuvant. The resulting antisera are testedfor antipeptide activity, for example, by binding the peptide toplastic, blocking with 1% BSA, reacting with rabbit antisera, washing,and reacting with radioiodinated, goat anti-rabbit IgG.

[0218] XI Purification of Naturally Occurring HPIP Using SpecificAntibodies

[0219] Naturally occurring or recombinant HPIP is substantially purifiedby immunoaffinity chromatography using antibodies specific for HPIP. Animmunoaffinity column is constructed by covalently coupling HPIPantibody to an activated chromatographic resin, such as CnBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0220] Media containing HPIP is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of HPIP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HPIP binding (e.g., a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and HPIP is collected.

[0221] XII Identification of Molecules which Interact with HPIP

[0222] HPIP or biologically active fragments thereof are labeled with1251 Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled HPIP, washed and any wells withlabeled HPIP complex are assayed. Data obtained using differentconcentrations of HPIP are used to calculate values for the number,affinity, and association of HPIP with the candidate molecules.

[0223] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 4 251 amino acids amino acid single linear Consensus 1813361 1 Met AspMet Lys Arg Arg Ile His Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 AlaAla Val Arg Glu Leu Val Leu Asp Asn Cys Lys Ser Asn Asp Gly 20 25 30 LysIle Glu Gly Leu Thr Ala Glu Phe Val Asn Leu Glu Phe Leu Ser 35 40 45 LeuIle Asn Val Gly Leu Ile Ser Val Ser Asn Leu Pro Lys Leu Pro 50 55 60 LysLeu Lys Lys Leu Glu Leu Ser Glu Asn Arg Ile Phe Gly Gly Leu 65 70 75 80Asp Met Leu Ala Glu Lys Leu Pro Asn Leu Thr His Leu Asn Leu Ser 85 90 95Gly Asn Lys Leu Xaa Asp Ile Ser Thr Leu Glu Pro Leu Lys Lys Leu 100 105110 Glu Cys Leu Lys Ser Leu Asp Leu Phe Asn Cys Glu Val Thr Asn Leu 115120 125 Asn Asp Tyr Arg Glu Ser Val Phe Lys Leu Leu Pro Gln Leu Thr Tyr130 135 140 Leu Asp Gly Tyr Asp Arg Glu Asp Gln Glu Ala Pro Asp Ser AspAla 145 150 155 160 Glu Val Asp Gly Val Asp Xaa Xaa Glu Glu Asp Gly GluGly Glu Asp 165 170 175 Glu Glu Asp Glu Asp Asp Glu Asp Gly Glu Glu GluGlu Phe Asp Glu 180 185 190 Glu Asp Asp Glu Asp Glu Asp Val Glu Gly AspGlu Asp Asp Asp Glu 195 200 205 Val Ser Glu Glu Glu Glu Glu Phe Gly LeuAsp Glu Glu Asp Glu Asp 210 215 220 Glu Asp Glu Asp Glu Glu Glu Glu GluGly Gly Lys Gly Glu Lys Arg 225 230 235 240 Lys Arg Glu Thr Asp Asp GluGly Glu Asp Asp 245 250 966 base pairs nucleic acid single linearConsensus 1813361 2 GACGGCCCTC GCTGCGCAAG CGGGGACGNC TNTNCCCCCTNCGACCCCGC CGCGGGAAAG 60 TTAAGTTTGA AGAGGGGGGA AGAGGGGAAC ATGGACATGAAGAGGAGGAT CCACCTGGAG 120 CTGAGGAACC GGACCCCGGC AGCTGTTCGA GAACTTGTCTTGGACAATTG CAAATCAAAT 180 GATGGAAAAA TTGAGGGCTT AACAGCTGAA TTTGTGAACTTAGAGTTCCT CAGTTTAATA 240 AATGTAGGCT TGATCTCAGT TTCAAATCTC CCCAAGCTGCCTAAATTGAA AAAGCTTGAA 300 CTCAGTGAAA ATAGAATCTT TGGAGGTCTG GACATGTTAGCTGAAAAACT TCCAAATCTC 360 ACACATCTAA ACTTAAGTGG AAATAAACTG ANAGATATCAGCACCTTGGA ACCTTTGAAA 420 AAGTTAGAAT GTCTGAAAAG CCTGGACCTC TTTAACTGTGAGGTTACCAA CCTGAATGAC 480 TACCGAGAGA GTGTCTTCAA GCTCCTGCCC CAGCTTACCTACTTGGATGG CTATGACCGA 540 GAGGACCAGG AAGCACCTGA CTCAGATGCC GAGGTGGATGGTGTTGATNA AGANGAGGAG 600 GACGGAGAAG GAGAAGATGA GGAAGACGAG GACGATGAGGATGGTGAAGA AGAGGAGTTT 660 GATGAAGAAG ATGATGAAGA TGAAGATGTA GAAGGGGATGAGGACGACGA TGAAGTCAGT 720 GAGGAGGAAG AAGAATTTGG ACTTGATGAA GAAGATGAAGATGAGGATGA GGATGAAGAG 780 GAGGAAGAAG GTGGGAAAGG TGAAAAGAGG AAGAGAGAAACAGATGATGA AGGAGAAGAT 840 GATTAAGACC CCAGATGACC TGCAGAAACA GAACTTTTCAGTATTGGTTG GACTGCTCAT 900 GGATTTNNTA GCTGTTTAAA AAAAAACCCC CNCTAGCTGTGNTNCAACCC CCCAGGCCAC 960 CCCACC 966 251 amino acids amino acid singlelinear GenBank 1498225 3 Met Asp Met Lys Arg Arg Ile His Leu Glu Leu ArgAsn Arg Thr Pro 1 5 10 15 Ala Ala Val Arg Glu Leu Val Leu Asp Asn CysLys Ser Asn Asp Gly 20 25 30 Lys Ile Glu Gly Leu Thr Ala Glu Phe Val AsnLeu Glu Phe Leu Ser 35 40 45 Leu Ile Asn Val Gly Leu Ile Ser Val Ser AsnLeu Pro Lys Leu Pro 50 55 60 Lys Leu Lys Lys Leu Glu Leu Ser Glu Asn ArgIle Phe Gly Gly Leu 65 70 75 80 Asp Met Leu Ala Glu Lys Leu Pro Asn LeuThr His Leu Asn Leu Ser 85 90 95 Gly Asn Lys Leu Lys Asp Ile Ser Thr LeuGlu Pro Leu Lys Lys Leu 100 105 110 Glu Cys Leu Lys Ser Leu Asp Leu PheAsn Cys Glu Val Thr Asn Leu 115 120 125 Asn Asp Tyr Arg Glu Ser Val PheLys Leu Leu Pro Gln Leu Thr Tyr 130 135 140 Leu Asp Gly Tyr Asp Arg GluAsp Gln Glu Ala Pro Asp Ser Asp Ala 145 150 155 160 Glu Val Asp Gly ValAsp Glu Glu Glu Glu Asp Glu Glu Gly Glu Asp 165 170 175 Glu Glu Asp GluAsp Asp Glu Asp Gly Glu Glu Glu Glu Phe Asp Glu 180 185 190 Glu Asp AspGlu Asp Glu Asp Val Glu Gly Asp Glu Asp Asp Asp Glu 195 200 205 Val SerGlu Glu Glu Glu Glu Phe Gly Leu Asp Glu Glu Asp Glu Asp 210 215 220 GluAsp Glu Asp Glu Glu Glu Glu Glu Gly Gly Lys Gly Glu Lys Arg 225 230 235240 Lys Arg Glu Thr Asp Asp Glu Gly Glu Asp Asp 245 250 249 amino acidsamino acid single linear GenBank 403007 4 Met Glu Met Gly Arg Arg IleHis Leu Glu Leu Arg Asn Arg Thr Pro 1 5 10 15 Ser Asp Val Lys Glu LeuVal Leu Asp Asn Ser Arg Ser Asn Glu Gly 20 25 30 Lys Leu Glu Gly Leu ThrAsp Glu Phe Glu Glu Leu Glu Phe Leu Ser 35 40 45 Thr Ile Asn Val Gly LeuThr Ser Ile Ala Asn Leu Pro Lys Leu Asn 50 55 60 Lys Leu Lys Lys Leu GluLeu Ser Asp Asn Arg Val Ser Gly Gly Leu 65 70 75 80 Glu Val Leu Ala GluLys Cys Pro Asn Leu Thr His Leu Asn Leu Ser 85 90 95 Gly Asn Lys Ile LysAsp Leu Ser Thr Ile Glu Pro Leu Lys Lys Leu 100 105 110 Glu Asn Leu LysSer Leu Asp Leu Phe Asn Cys Glu Val Thr Asn Leu 115 120 125 Asn Asp TyrArg Glu Asn Val Phe Lys Leu Leu Pro Gln Leu Thr Tyr 130 135 140 Leu AspGly Tyr Asp Arg Asp Asp Lys Glu Ala Pro Asp Ser Asp Ala 145 150 155 160Glu Gly Tyr Val Glu Gly Leu Asp Asp Glu Glu Glu Asp Glu Asp Glu 165 170175 Glu Glu Tyr Asp Glu Asp Ala Gln Val Val Glu Asp Glu Glu Asp Glu 180185 190 Asp Glu Glu Glu Glu Gly Glu Glu Glu Asp Val Ser Gly Glu Glu Glu195 200 205 Glu Asp Glu Glu Gly Tyr Asn Asp Gly Glu Val Asp Asp Glu GluAsp 210 215 220 Glu Glu Glu Leu Gly Glu Glu Glu Arg Gly Gln Lys Arg LysArg Glu 225 230 235 240 Pro Glu Asp Glu Gly Glu Asp Asp Asp 245

What is claimed is:
 1. A substantially purified human phosphataseinhibitor protein (HPIP) comprising the amino acid sequence of SEQ IDNO:1 or fragments thereof.
 2. An isolated and purified polynucleotidesequence encoding the HPIP of claim
 1. 3. A polynucleotide sequencewhich hybridizes under stringent conditions to the polynucleotidesequence of claim
 2. 4. A hybridization probe comprising thepolynucleotide sequence of claim
 2. 5. An isolated and purifiedpolynucleotide sequence comprising SEQ ID NO:2 or variants thereof.
 6. Apolynucleotide sequence which is complementary to SEQ ID NO:2 orvariants thereof.
 7. A hybridization probe comprising the polynucleotidesequence of claim
 6. 8. An expression vector containing thepolynucleotide sequence of claim
 2. 9. A host cell containing the vectorof claim
 8. 10. A method for producing a polypeptide comprising theamino acid sequence of SEQ ID NO:1 the method comprising the steps of:a) culturing the host cell of claim 9 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.
 11. A pharmaceutical composition comprising asubstantially purified HPIP having an amino acid sequence of SEQ ID NO:1in conjunction with a suitable pharmaceutical carrier.
 12. A purifiedantibody which binds specifically to the polypeptide of claim
 1. 13. Apurified agonist which specifically binds to and modulates the activityof the polypeptide of claim
 1. 14. A purified antagonist whichspecifically binds to and modulates the activity of the polypeptide ofclaim
 1. 15. A method for treating diseases resulting from excessivecell growth and division comprising administering to a subject in needof such treatment an effective amount of the agonist of claim
 13. 16. Amethod for treating diseases resulting from insufficient cell growth anddivision comprising administering to a subject in need of such treatmentan effective amount of the antagonist of claim
 14. 17. A method fordetection of polynucleotides encoding HPIP in a biological samplecomprising the steps of: a) hybridizing a polynucleotide consisting ofthe polynucleotide of claim 6 to nucleic acid material of a biologicalsample, thereby forming a hybridization complex; and b) detecting saidhybridization complex, wherein the presence of said complex correlateswith the presence of a polynucleotide encoding HPIP in said biologicalsample.
 18. The method of claim 17 wherein before hybridization, thenucleic acid material of the biological sample is amplified by thepolymerase chain reaction.
 19. A method for treating a disease orcondition associated with decreased expression of functional HPIP,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional HPIP, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional HPIP, comprising administering to a patientin need of such treatment a composition of claim
 24. 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, the method comprising: a) combining the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 27. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, the methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of HPIP in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of HPIP in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofHPIP in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptide havingthe amino acid sequence of SEQ ID NO:1, or an immunogenic fragmentthereof, under conditions to elicit an antibody response, b) isolatingantibodies from said animal, and c) screening the isolated antibodieswith the polypeptide, thereby identifying a polyclonal antibody whichbinds specifically to a polypeptide having the amino acid sequence ofSEQ ID NO:1.
 37. A polyclonal antibody produced by a method of claim 36.38. A composition comprising the polyclonal antibody of claim 37 and asuitable carrier.
 39. A method of making a monoclonal antibody with thespecificity of the antibody of claim 11, the method comprising: a)immunizing an animal with a polypeptide having the amino acid sequenceof SEQ ID NO:1, or an immunogenic fragment thereof, under conditions toelicit an antibody response, b) isolating antibody producing cells fromthe animal, c) fusing the antibody producing cells with immortalizedcells to form monoclonal antibody-producing hybridoma cells, d)culturing the hybridoma cells, and e) isolating from the culturemonoclonal antibody which binds specifically to a polypeptide having theamino acid sequence of SEQ ID NO:1.
 40. A monoclonal antibody producedby a method of claim
 39. 41. A composition comprising the monoclonalantibody of claim 40 and a suitable carrier.
 42. The antibody of claim11, wherein the antibody is produced by screening a Fab expressionlibrary.
 43. The antibody of claim 11, wherein the antibody is producedby screening a recombinant immunoglobulin library.
 44. A method ofdetecting a polypeptide having the amino acid sequence of SEQ ID NO:1 ina sample, the method comprising: a) incubating the antibody of claim 11with a sample under conditions to allow specific binding of the antibodyand the polypeptide, and b) detecting specific binding, wherein specificbinding indicates the presence of a polypeptide having the amino acidsequence of SEQ ID NO:1 in the sample.
 45. A method of purifying apolypeptide having the amino acid sequence of SEQ ID NO:1 from a sample,the method comprising: a) incubating the antibody of claim 11 with asample under conditions to allow specific binding of the antibody andthe polypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide having the amino acid sequence of SEQID NO:1.
 46. A microarray wherein at least one element of the microarrayis a polynucleotide of claim
 13. 47. A method of generating a transcriptimage of a sample which contains polynucleotides, the method comprising:a) labeling the polynucleotides of the sample, b) contacting theelements of the microarray of claim 46 with the labeled polynucleotidesof the sample under conditions suitable for the formation of ahybridization complex, and c) quantifying the expression of thepolynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO:2.